Virulent Brucella abortus Prevents Lysosome Fusion and Is Distributed within Autophagosome-Like Compartments

ABSTRACT Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting thatBrucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.

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Brucella abortus Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes

Infection and Immunity ◽

10.1128/iai.66.12.5711-5724.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 5711-5724 ◽

Cited By ~ 284

Author(s):

Javier Pizarro-Cerdá ◽

Stéphane Méresse ◽

Robert G. Parton ◽

Gisou van der Goot ◽

Alberto Sola-Landa ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Brucella Abortus ◽

Cathepsin D ◽

Bacterial Strains ◽

Infected Cells ◽

Bacterial Replication ◽

Protein Disulfide ◽

Intracellular Pathway ◽

Replication Compartment

ABSTRACT Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.

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Brucella abortus efp gene is required for an efficient internalization in HeLa cells

Microbial Pathogenesis ◽

10.1016/j.micpath.2011.09.008 ◽

2012 ◽

Vol 52 (1) ◽

pp. 31-40 ◽

Cited By ~ 13

Author(s):

Florencia Iannino ◽

Juan E. Ugalde ◽

Nora Iñón de Iannino

Keyword(s):

Hela Cells ◽

Brucella Abortus

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Stable transfection of the estrogen receptor cDNA into Hela cells induces estrogen responsiveness of endogenous cathepsin D gene but not of cell growth

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(90)91440-4 ◽

1990 ◽

Vol 169 (1) ◽

pp. 109-115 ◽

Cited By ~ 25

Author(s):

Isabelle Touitou ◽

Marc Mathieu ◽

Henri Rochefort

Keyword(s):

Estrogen Receptor ◽

Cell Growth ◽

Hela Cells ◽

Cathepsin D ◽

Stable Transfection ◽

Receptor Cdna

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A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells

Microbiology ◽

10.1099/mic.0.060509-0 ◽

2012 ◽

Vol 158 (10) ◽

pp. 2610-2618 ◽

Cited By ~ 5

Author(s):

Marie de Barsy ◽

Aurélie Mirabella ◽

Jean-Jacques Letesson ◽

Xavier De Bolle

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Brucella Abortus

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Survival of Tropheryma whipplei, the Agent of Whipple's Disease, Requires Phagosome Acidification

Infection and Immunity ◽

10.1128/iai.70.3.1501-1506.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1501-1506 ◽

Cited By ~ 65

Author(s):

Eric Ghigo ◽

Christian Capo ◽

Marianne Aurouze ◽

Ching-Hsuan Tung ◽

Jean-Pierre Gorvel ◽

...

Keyword(s):

Hela Cells ◽

Cathepsin D ◽

Tropheryma Whipplei ◽

Host Cells ◽

Whipple’S Disease ◽

Acidic Ph ◽

Bafilomycin A1 ◽

Phagosome Maturation ◽

Whipple's Disease ◽

New Approach

ABSTRACT Tropheryma whipplei was established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here that T. whipplei survives within HeLa cells by controlling the biogenesis of its phagosome. Indeed, T. whipplei colocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition, T. whipplei survived within HeLa cells by adapting to acidic pH. The vacuoles containing T. whipplei were acidic (pH 4.7 ± 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride, N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing of T. whipplei. The ability of T. whipplei to survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.

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FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION

10.1101/2021.02.23.432539 ◽

2021 ◽

Author(s):

Cristina Escrevente ◽

Ana S. Falcão ◽

Michael J. Hall ◽

Mafalda Lopes-da-Silva ◽

Pedro Antas ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Cathepsin D ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Single Pulse ◽

Photoreceptor Outer Segments ◽

Lysosomal Ph ◽

Electron Microscopical ◽

Phagosome Fusion

AbstractPurposeWe aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers.MethodsWe fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24h the cells started accumulating AFGs similar to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking Cathepsin D by gene editing.ResultsAFGs appear to derive from incompletely digested POS-containing phagosomes and are surrounded after 72h by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF accumulation.ConclusionsWe conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.

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Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.6.3020-3027.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3020-3027 ◽

Cited By ~ 63

Author(s):

Suk Kim ◽

Masahisa Watarai ◽

Yuki Kondo ◽

Janchivdorj Erdenebaatar ◽

Sou-ichi Makino ◽

...

Keyword(s):

Hela Cells ◽

Brucella Abortus ◽

Kanamycin Resistance ◽

Host Cells ◽

Class Iii ◽

Wild Type ◽

Intracellular Growth ◽

Isolation And Characterization ◽

Bacterial Genes

ABSTRACT Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

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The catalytically inactive precursor of cathepsin D induces apoptosis in human fibroblasts and HeLa cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.21269 ◽

2007 ◽

Vol 101 (6) ◽

pp. 1558-1566 ◽

Cited By ~ 20

Author(s):

Olga Schestkowa ◽

Dominik Geisel ◽

Ralf Jacob ◽

Andrej Hasilik

Keyword(s):

Hela Cells ◽

Cathepsin D ◽

Human Fibroblasts

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Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

Infection and Immunity ◽

10.1128/iai.69.7.4528-4535.2001 ◽

2001 ◽

Vol 69 (7) ◽

pp. 4528-4535 ◽

Cited By ~ 91

Author(s):

Gabriel Briones ◽

Nora Iñón de Iannino ◽

Mara Roset ◽

Ana Vigliocco ◽

Patricia Silva Paulo ◽

...

Keyword(s):

Hela Cells ◽

Brucella Abortus ◽

Virulent Strain ◽

Sodium Dodecyl ◽

Wild Type ◽

Log Reduction ◽

Vaccinal Strain ◽

Intracellular Multiplication ◽

Glucan Synthetase ◽

Residual Virulence

ABSTRACT Null cyclic β-1,2-glucan synthetase mutants (cgsmutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuatedB. abortus S19 strain. Although the cgsmutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected withB. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by thecgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

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